Staphylococcus aureus is one of the most common causes of acute and chronic infections in both community and hospital settings. The ability of S. aureus to adhere to host tissues or medical implants and develop into biofilms is a critical determinant of chronic infections. The challenge presented by a biofilm is its extraordinary resistance to both host defenses and antimicrobial therapies. We recently demonstrated that the S. aureus cell-to-cell communication system, also called quorum-sensing or the agr system, controls the switch between the planktonic and biofilm state. We speculate that other regulatory pathways feed into this control mechanism. We have begun screening a mariner transposon library and discovered that insertions in the rsbUVW-sigB locus eliminated the ability of S aureus to form a biofilm. The phenotype was due to lack of Sigma B (SigB) activity and extended to community-associated methicillin-resistant S. aureus (CA-MRSA) strains. Our central hypothesis is that an increased level of extracellular enzymes blocks biofilm formation in SigB defective strains. For Specific Aim 1, we will (i) define the extracellular proteome of SigB defective strains;(ii) characterize the role of identified protein(s) in the SigB biofilm phenotype;and (iii) investigate the role of SigB in biofilm dispersal. We previously demonstrated that increased agr expression has anti-biofilm properties, and it is evident from our studies and published reports that defects in the SigB system upregulate agr expression. In support of this finding, we discovered that mutations in the agr system restore biofilm formation in SigB-defective strains. We hypothesize that the SigB system is controlling the expression of an unknown protein that affects agr. To address the interconnection of these regulatory cascades, in Specific Aim 2, we will (i) determine whether the ArlRS two-component system could be the unknown protein;(ii) employ a proteomic approach to identify proteins bound to the agr promoter;and (iii) characterize the identified protein(s). In both specific aims, we will repeat experiments in a CA-MRSA strain to gain insight on the contribution of SigB to the exceptionally virulent nature of these isolates. Altogether, the proposed experiments will define the role of SigB in biofilm formation and the interconnection with the agr quorum-sensing system. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a leading cause of chronic bacterial disease. Our preliminary studies have demonstrated that the Sigma B regulatory cascade in methicillin-susceptible and methicillin-resistant S. aureus (MRSA) is essential for biofilm formation, a known determinant of chronic infections. An improved understanding of role of Sigma B in biofilm formation could aid the development of innovative treatment options for eliminating S. aureus biofilms.